抗氧化物的测定2

丙二醛MDA的测定(1) 0.05-0.1g叶片液氮研磨后，加入ImL 0.1%TCA,摇晃均匀，加入lOOyL 5mg/mL的丁羟甲苯(已有，在95度水浴中温浴30分钟。然后15000g离心10min, 0.55mL的上清液中加入0.55mL TBA(25%的硫代巴比土酸)，然后再在95度水 浴中温浴30分钟，在冰上冷却，10000g离心5min，将上清液在532nm测定吸 光度，通过扣除600nm下吸收值进行矫正，根据MDA标准曲线定其含量。The level of lipid peroxidation in leaf tissues was determined in terms of the peroxidation byproduct MDA in the samples. Briefly, 50 to 100 mg leaf tissuewas shock frozen in liquid nitrogen and ground in 1mL of 0.1% TCA. To the homogenized samples, 0.1 mL butylated hydroxytoluene (5 mg/mL) was added and the homogenates were incubated at 95_C for 30 min, centrifuged at 15,000g for 10 min, and 0.55 mL of the supernatant fraction was mixed with 0.55 mL of TBA solution (25% thiobarbituric acid). The mixture was heated at 95_C for 30 min, chilled on ice, and then centrifuged at 10,000g for 5 min. The absorbance of the supernatant was measured at 532 nm and corrected by subtracting nonspecific absorption at 600 nm. The amount of MDA was calculated according to an MDA standard curve. From: Specific Roles of a- and g-Tocopherol in Abiotic Stress responses of Transgenic Tobacco(2) 0.1g组织浸在2mL 0.1%TCA(三氯乙酸)中，匀浆10400rpm 离心5min,每 200yL上清液加入800yL 20%的TCA(包含5% TBA)，混合物在95度温浴30min, 迅速在冰上冷却，然后混合物在10400rpm离心15min，上清液在532nm测定 吸光度。扣除600nm下吸收值进行矫正，用消光系数155Mm-1cm-1计算MDA值。Approximately 100 mg of tissue was macerated in 2 ml of 0.1% trichloroacetic acid. The homogenate was centrifuged at 10,400 rpm for 5 min. For every 200“1 of the aliquot of the supernatant, 800 “l of 20% TCA containing 0.5% TBA was added. The mixture was heated at 95 C for 30 min and then cooled quickly in an ice bath. The resulting mixture was centrifuged at 10,400 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. The concentration of malondialdehyde content was calculated by using the extinction coefficient of 155 mM-l cm-l.From: Increased -tocopherol content in soybean seed overexpressingthe Perilla frutescens y -tocopherol methyltransferase gene(3)称取剪碎的试材1 g,加入2 mL 10%TCA，研磨至匀浆，再加8 mLTCA进 -步研磨，匀浆4000 rp m离心10 min，上清液为样品提取液。吸取离心的上清液1.5 mL (对照加1.5 ml蒸馏水)，加入2.5 mL 0.5%TBA溶 液，混匀物于沸水浴上反应15 min,迅速冷却后，12000 rpm离心5 min。取上清 液在532 nm、600 nm和450 nm波长下测定OD值。利用公式：C (ymol/L) =6.45x(D532 - D600) - 0.56xD450计算 MDA 浓度。计算根据植物组织的重量计算测定样品中MDA的含量：MDA (卩mol/gFW)=CxV/W式中C-MDA浓度(yM)； V 提取液总体积(mL)； W植物组织鲜重(g)。(拟南芥生育酚环化酶基因(VTE1)遗传转化和转基因烟草 抗性的研究 )文献 Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana 中用到的方法与此类似,它 参考的文献： Hodges DM, DeLong JM, Forney CF et al (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604-611 在网上查不到原文维生素C的测定(1) 2平方厘米的叶片在液氮中磨碎，加入1.1mL5%的磺基水杨酸，离心 后上清液与等体积的150mM NaH2PO4(pH 7.4)稀释，冰上静置，用10N的NaOH 调pH至5.5-6.5，为了测定还原的维生素C，200吐的提取物连续的与100吐的 双蒸水、200吐 的10%的TCA、200吐 的44%的H3PO4、200吐 的4%的联吡啶 (溶于70%的乙醇)、100吐 的3%的FeCL3,混合物在60度温浴30min, 525nm 下检测吸光度，用标准曲线测定浓度(该方法可用)加入5010 mmol / L DTT(二硫苏糖醇)，25 C保温10 min，使DHA还原为ASA，此法用来测定样品中总的抗坏血酸的量。在还原型抗坏血酸(ASA)的测 定中，只要把上述的DTT和乙基马来酰亚胺用等体积的蒸馏水替代即可。以磺 基水杨酸为溶剂，用同样的方法制作ASA标准曲线。Tobacco leaf discs (2 cm2) were ground in liquid N2 to a fine powder and samples were extracted with 1.1 mL 5% (w/v) 5-sulfosalicylic acid. After centrifugation the supernatant was diluted 1:1 with 150 mM NaH2PO4 buffer (pH 7.4) and stored on ice. The pH was adjusted to 5.5 to 6.5 using 10 N NaOH. For determination of reduced ascorbate, 200 mL of the extract was successively mixed with 100 mL double distilled water, 200 mL 10% (w/v) TCA, 200 mL 44% (v/v) H3PO4,200 mL 4% (w/v) 2,2#-dipyridyl dissolved in 70% (v/v) ethanol, and 100 mL 3% (w/v) FeCl3. The mixtures were incubated for 60 min at 30_C and finally the color formation was measured at 525 nm. The ascorbate content was calculated using a standard curve measured with freshly prepared ascorbate standards. For determination of total ascorbate, the neutralized extracts were oxidized by adding 50 mL dithiothreitol (10 mM) to the samples instead of distilled water.After 15 min incubation at room temperature, 50 mL 0.5% (w/v) N-ethylmaleimide were added followed by the same procedure as described above.From: Specific Roles of a- andg-Tocopherol in Abiotic Stress responses of Transgenic Tobacco(2) lcm2叶片组织磨碎，浸入ImL 6%的HCLO4，粗提物2度lOOOOg离 心lOmin,上清收集用于分析，加NaCO3调pH增加1个单位。维生素C在0.2mM 乙酸钠缓冲液(pH 5.6)，在室温和5个单位的维生素C氧化酶反应15min.在 265nm 下测定。标准品做标准曲线测浓度。Ascorbate content was measured according to the method of Foyer et al. (1989). Leaf tissue (1 cm) was ground in a prechilled mortar with acid-washed sand in 1 mL of 6% perchloric acid. The crude extract was centrifuged at 2C for 1O min at 1O,OOOg and the supernatant was collected for analysis. The pH of the extract was increased to approximately 1 by stepwise addition of saturated Na,CO,. Ascorbate was assayed spectrophotometrically at 265 nm in O.2 M sodium acetate buffer, pH 5.6, before and after a 15-min incubation with 5 units of ascorbate oxidase (from Curcurbita, Sigma) at room temperature. Ascorbate levels were determined from a standard curve. From: Acclimation of Foliar Antioxidant Systems to Growth Irradiance in Three Broad-Leaved Evergreen Species文中所参考的文献：Foyer CH, Dujardyn M, Lemoine Y(1989) Responses of photosynthesis and the xanthophyll and ascorbate-glutathione cycles to changes in irradiance, photoinhibition and recovery.Plant Physiol Biochem 27: 751-76O (网上查不到 原文)谷胱甘肽的测定(1)0.05g叶片液氮磨碎，加入0.1M HCL1mL，冰上15min,每2min涡流 一次，20000g 5min离心两次，上清转移到新管。50yL上清用等体积的90mM NaOH中和，加溴甲烷在 黑暗下反应。为了测定总的谷胱甘肽含量，20吐1M Tris-HCL Ph 8.0、10pL10mM DDT、加入到中和的提取物里总计270吐，样品室 温 黑暗条件下60min,完全还原谷胱甘肽，25吐 溴甲烷(溶于乙腈10mM)，室 温黑暗15min，加入705yL乙酸中止反应。为了测定氧化谷胱甘肽，中和的样品加入20吐1M Tris-HCL pH 8.0、10吐 5mM的NEM总体积260yL,室温10min,再加入10mM DDT，后同上。在380nm 激发光后用480nm荧光检测 乙酸钾溶液(pH 5.5)和甲醇作为流动相All solutions used during extraction and derivatization procedure were bubbled with nitrogen for 2 min to remove oxygen from the aqueous phase. Deep-frozen leaf punches of 50 mg fresh weight were homogenized in liquid nitrogen to a fine powder and extracted with 1 mL 0.1 m hydrochloric acid (HCl). The homogenates were incubated on ice for 15 min and vortexed every 2 min.The extracts were cleared by two consecutive centrifugations for 5 min at 20 000 g, after which the supernatant was transferred to fresh tubes, respectively.Prior to the measurement, 50 mL aliquots of the cleared extracts were neutralized with an equal volume of 90 mm NaOH and derivatized with monobromobimane in dark tubes as follows.For the determination of total glutathione, 20 mL 1 m Tris/ HCl, pH 8.0, and 10 mL 10mm DTT were added to the neutralized extracts in a total volume of 270 mL, and the samples were incubated at room temperature in the dark for 60 min to completely reduce the glutathione pool. For derivatization, 25 mL monobromobimane (10 mm in acetonitrile) was added and the samples were incubated for 15 min in the dark at room temperature before the reactions were stopped by adding 705 mL 5% (v/v) acetic acid.For the determination of oxidized glutathione, the neutralized samples were incubated with 20 mL 1 m Tris/HCl, pH 8.0, and 10 mL 5mm NEM in a total volume of 260 mL for 10 min at room temperature prior to the addition of 10 mL 10mm DTT and further processing as described for the determination of total glutathione.Total and oxidized glutathione were detected by fluorescence at 480 nm after excitation at 380 nm and quantified in comparison to standard solutions after isocratic separation at a flow rate of 1 mL min-1 and a temperature of 37 C on a Phenomenex (Aschaffenburg, Germany) Luna 5u-C18 reverse phase column (4.6 250 mm) on a Dionex (Idstein, Germany) Summit HPLC system, using eluents A (100 mm potassium acetate, pH 5.5) and B (methanol) applying the following gradient: 11% eluent A and 89% eluent B at 0-19 min, 100% eluent B at 19-26 min prior to reequilibration with 11% eluent A/89% eluent B for 10 min. from: Tocopherol deficiency in transgenic tobacco (Nicotiana tabacuLm.) plants leads to accelerated senescence(2) Lcm2叶片组织磨碎，浸入1mL 7%磺基水杨酸，412nm下测定吸光度, 一部分提取物30倍稀释于缓冲液(0.5M KH2PO4、6.3mM EDTA, pH 7.6)，接着 加入1.2mM 2-硝基苯甲酸和0.2mM NADPH，加入0.2单位的谷胱甘肽还原酶开 始反应，总体积2 mL, GSH标准做曲线。(该方法可用)Leaf tissue (1 cm) was ground in a prechilled mortar with acid-washed sand in 1 mL of 7% sulfosalicylic acid。Total glutathione was determined spectrophotometrically at 412 nm by the cycling method described by Griffith (1980). A portion of the extract was neutralized by a 30-fold dilution in 0.5 M KH,PO, 6.3 mM EDTA, pH 7.6, and subsequently assayed in the presence of 1.2 mM 5,5- dithiobis-(2-nitrobenzoic acid) and 0.2 mM NADPH. The reaction was started by the addition,of 0.2 unit of GR (from yeast, Boehringer Mannheim) in a total volume of 2 mL. A11 values are expressed as GSH equivalents, determined from a standard curve. From: Acclimation of Foliar Antioxidant Systems to Growth Irradiance in Three Broad-Leaved Evergreen Species文中所参考的文献：determination of glutathione and glutathione disulfide in biological samples. Griffith OW . 1980 (网上查不到原文)SOD活性的测定1.酶液制备 取1.0 g叶片剪碎，置入玻璃匀浆杯中，加入预冷的20 mM KH2PO45 mL进行冰浴研磨提取。匀浆液低温12000 rpm离心15 min,取上清液4 C保存备 用。2. 酶活性测定以不加酶液（用缓冲液代替）的试管为最大光化还原管,用缓冲 液作空白管。各管在4000 lux、25 C反应20 min进行光化还原，置暗处终止，立 即测反应液的O D560值。3. 计算以抑制NBT光化还原50%作为一个酶单位（n），酶活性以n/mg蛋白表示。n =（ODmax-OD560） /ODmax/2 （拟南芥生育酚环化酶基因（VTE1）遗传转化和转基因烟草抗性的研究 ）POD活性测定1. 酶液制备同上。2. 酶活性测定 取20吐酶液（用PBS代替酶液作空白）加30 mL反应混合液， 混匀，25 C温浴5 min,口20吐H2O2启动反应于470 nm波长处作时间扫描，开 始记录数据。3. 计算以每分钟OD470的变化值0.01为一个相对酶活单位，计算叶片内过 氧化物酶酶活力的大小（单位：U/g鲜重）。（拟南芥生育酚环化酶基因（VTE1）遗 传转化和转基因烟草抗性的研究 ）APX 酶活性在 340nm 下分光光度测定，分析培养基（ 50Mm Hepes-KOH、 0.1mM EDTA、0.5 mM 维生素 C，pH 7.6），加入 0.2 mM 的 H2O2 开始反应，活 性矫正通过无样品时H2O2维生素C的氧化作用，每摩尔消光系数2800cm-1用 于计算酶活性GR酶活性在340nm下测定，分析培养基（100mM 的Tris-HCL、1mM EDTA、 0.5mM GSSG, pH 8.0），加入0.05mM NADPH 开始反应。扣除无GSSG的非特 异性 NADPH 氧化酶活性速率，每摩尔消光系数 6200cm-1 用于计算酶活性MDAR 活性在 340nm 下测定，分析培养基（ 50mM Hepes-KOH、 0.1mM EDTA、2.5 mM 维生素C、0.1mM NADH，pH 7.6），加入4个单位的维生素C 氧化酶开始反应，产生单脱水抗坏血酸基。扣除无样品的非特异性NADH氧化 酶活性速率可溶性糖、淀粉和游离氨基酸的测定（1） 碎的叶片在80%的乙醇里提取，微滴定的方法测定可溶性糖和淀粉，通 过在340nm下测定NADPH的吸收值来测定。用AccQ Taq衍生一级和二级氨基 酸，HPLC分析（另见参考），溶液A （140mM 的乙酸钠pH 5.8 , 7 mM的三 乙醇胺）、B乙腈、C水 荧光检测（激发光250 nm 检测光395 nm）Shock-frozen leaf samples were extracted in 80% ethanol and assayed for soluble sugars and starch as described by( Stitt etal., 1989) using a microtiter plate reader. For the determination of amino acid contents, primary and secondary amino acids were derivatized using the fluoropohore 6-aminoquinolyl- N-hydroxysuccimidyl carbamate (AccQ Taq) and separated at a flow rate of 1 mL/min at 37_C on a Dionex Summit HPLC system essentially as described by van Wandelen and Cohen (1997), using the eluents A (140 mM sodium acetate, pH 5.8; 7 mM triethanolamine), B (acetonitrile), and C (water) and fluorescence detection (excitation at 250 nm and detection at 395 nm). From: Specific Roles of a- and g-Tocopherol in Abiotic Stress responses of Transgenic Tobacco(2)叶片用 80%的乙醇(10mM Hepes-KOH, pH 7.4)在 80oC 提取 1-2 小时， 上清液用于葡萄糖、果糖、蔗糖的测定。残留物二次提取。用于淀粉的测定。Leaf discs (taken at the times indicated) and potato tuber slices were extracted with 80% ethanol (10 mM HEPES-KOH, pH 7.4) at 80C for 1-2 h. The supernatant was used for the determination of glucose, fructose and sucrose (Stitt etal., 1989). The remainder was extracted a second time, washed in water and dried. Determination of starch was done as described by Stitt et a/. (1978). From: Expression ofE. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning 文中所参考的文献 ：测糖含量：Metabolite levels in specific cells and subcellular compartments of plant leaves. Stitt, M., Lilley, R.Mc.C., Gerhardt, R. and Heldt, H.W. Methods Enzymol.测淀粉含量： Pathway of starch breakdown in photosynthetic tissue of fisum sativum. Biochim. Stitt, M., Wirtz, W. and Heldt, H.W. Biophys. Acta, 544, 200-214.(两篇文献太久远 网上查不到)离子泄漏的测定(1)测定膜的损伤程度。每次测量，六片直径10mm的叶片放在8mL双蒸 水上漂浮，4度20个小时，用电导率仪测定液体的电导率，得到A值。然后将 叶片放回液体中，放入一封闭管中， 95 度保温 35min, 测电导率，得到 B 值。 A/B X100.Membrane damage was assayed by measuring ion leakage from leaf discs according to Rizhsky et al. (2002). For each measurement, six leaf discs (10mm diameter) were floated abaxial side up on 8 mL of double distilled water for 20 h at 4oC. Following incubation, the conductivity of the bathing solution was measured with a conductivity meter (value A). The leaf discs were then returned to the bathing solution, introduced into sealed tubes, and incubated with the bathing solution at 95_C for 30 min. After cooling to room temperature the conductivity of the bathingsolution was measured again (value B). For each measurement, ion leakage was expressed as percentage leakage, i.e. (value A/value B)_100.From: Specific Roles of a- and g-Tocopherolin Abiotic Stress responses of Transgenic Tobacco（2）直径 1cm 的叶片于 2mL 双蒸水室温置至少四个小时，测定电导率。然后 将提取液煮沸 30min, 冷却后再测定调导率，然后初始值比上最后的值就是电解 率Leaf discs 1 cm diam （10 discs of at least three plants from each line） were held under 2 ml deionized water at room temperature for at least 4 h. The conductivity was then measured. The tubes containing leaf discs were boiled for 30 min, cooled and the conductivity was then remeasured. Electrolyte leakage was calculated as the percentage of initial to final conductivity.From : Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana脯氨酸含量的测定取约0.5 g的叶片置于研钵中，室温下加入2 mL 3%磺基水杨酸充分研磨，匀 浆，7000 rpm离心10 min。2.取1 mL上清，加入1 mL冰醋酸及1 mL酸性茚三酮， 混匀。 3. 利用脯氨酸标样溶液绘制标准曲线。 4. 将所有样品管置沸水浴中1 h。 5.在样品管中加入2 mL甲苯，摇匀置非甲苯相无色。6.利用紫外可见分光光度 计，在520 nm处测样品的OD值。用甲苯作空白对照。7.根据标准曲线计算叶片 中脯氨酸含量。（拟南芥生育酚环化酶基因（VTE1 ）遗传转化和转基因烟草抗性的研究）